Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antigen, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
The analyte, which is the target of the assay is referred to herein as the ligand, and the labeled analyte is referred to as the labeled ligand (including immunocompetent derivatives and analogs of such ligand). Compounds which specifically recognize the ligand and the labeled ligand and react to form complexes with them are referred to herein as receptors. The receptor and the ligand or labeled ligand form a conjugate pair. Any member of the pair can function as a receptor or a ligand.
In competitive binding immunoassays, a labeled ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate receptor. Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
ligand+labeled ligand+receptor&lt;=&gt;ligand-receptor+labeled ligand-receptor.
In an alternative type of immunological assay, commonly referred to as a sandwich assay, a ligand (antibody or antigen analyte) is contacted with a receptor to cause the ligand to bind to the receptor. This complex is then contacted with a solution of a labeled binding agent, such as an antibody, which reacts with the bound ligand. The amount of bound labeled binding agent is thus directly proportional to the amount of bound ligand.
Dry immunoassay analytical elements are known. In general such elements comprise receptors, such as antibodies for a ligand, immobilized in a particulate layer. In addition the element usually contains a reagent system that through interaction with a bound or unbound species results in a signal that can be correlated to the concentration of ligand in a sample. In use the sample is manually combined with an enzyme labeled ligand and applied to the element. After a time a wash solution containing a substrate for the labeled ligand is applied to the particulate layer. The substrate is catalyzed by the enzyme label to form a reaction product that ultimately causes a signal, such as chemiluminescence, to develop that can be correlated to the concentration of the ligand in the sample. Signal development systems are known for other known conventional labels such as radioactive tags, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
One problem that occurs in immunoassays of serum samples is background interference caused by the presence of hemoglobin arising from red blood cell lysis. Hemoglobin is a pseudoperoxidase. As such it catalyzes oxidation of a leuco dye, or chemiluminescent compound precursors, similarly to peroxidase enzyme labels producing a background signal. This background signal causes assay results to lack precision and accuracy.
This problem is aggravated when detection and quantification at picomolar concentrations are required for many diagnostically important serum hormones and cancer markers. Among the medically important hormones present in serum at picomolar concentration are aldosterone, insulin, thyroid stimulating hormone (TSH), angiotensin, ocytocin, parathyroid hormone (PTH), growth hormone, adrenocorticotrophic hormone (ACTH), and vasopressin. To perform immunoassay for serum analytes present at picomolar concentration, a sensitive detection chemistry such as fluorescence or chemiluminescence is needed. Because of the sensitivity of this type of detection chemistry, background due to hemoglobin pseudoperoxidase activity, unbound label or chemical interferents present in the serum sample have to be thoroughly removed.